Non-Gelling Soluble Extracellular Matrix with Biological Activity

ABSTRACT

Provided are methods for preparing non-gelling, solubilized extracellular matrix (ECM) materials useful as cell growth substrates. Also provided are compositions prepared according to the methods as well as uses for the compositions. In one embodiment a device, such as a prosthesis, is provided which comprises an inorganic matrix into which the non-gelling, solubilized ECM composition is dispersed to facilitate in-growth of cells into the ECM and thus adaptation and/or attachment of the device to a patient. In another embodiment, the composition is delivered intraarticularly, intrathecally, intraoccularly, intracranially, and into pleural space.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/760,864, filed Mar. 16, 2018, which is the United States nationalphase of International Application No. PCT/US2016/052261 filed Sep. 16,2016, and claims the benefit of U.S. Provisional Patent Application No.62/220,409 filed Sep. 18, 2015, the disclosures of which are herebyincorporated in their entirety by reference.

BACKGROUND

Methods of preparation of extracellular matrix- (ECM-) derived gels aredescribed herein.

The use of extracellular matrix (ECM) scaffolds is commonplace as thesescaffolds have been shown to accommodate tissue remodeling in numeroustissues. Soluble forms of ECM, which form a hydrogel at body temperature(37° C.), are increasingly used in anatomic locations that do not permitthe use of an ECM scaffold. Similar to ECM scaffolds, the ECM hydrogelshave biological effects that include immune modulation and recruitmentof stem cells, among others. However, all therapeutic applications donot require a hydrogel and a non-gelling soluble form of ECM may bepreferred in many instances, e.g., intraarticular injections, enemasolutions, etc.

SUMMARY

Described herein is the preparation and biologic effects of anon-gelling soluble form of ECM. A solubilized ECM product, especiallyone that has not been dialyzed and/or crosslinked, has the ability toform a hydrogel when pH- and salt balanced, and warmed to 37° C. Manytherapeutic applications do not require or may preclude the use of ahydrogel. In such cases, a non-gelling soluble ECM may be preferred.

Solubilization of ECM can be achieved by enzymatic digestion (e.g.,pepsin). To ensure that the mixture is non-gelling, ECM is sterilized(e.g., by exposure to ethylene oxide, gamma-irradiation, or electronbeam irradiation), e.g., in solid sheet form or in comminuted powderform. The soluble form of ECM following these processing steps does notform a hydrogel—a typical feature of solubilized ECM—but retains thebiological effects that have implications in a wide range of potentialtherapeutics. Results of in-vitro studies show that the non-gellingsoluble ECM promotes the migration/chemotaxis of a multipotent stemcell, and stimulates macrophages in the same manner as an ECM hydrogel.

The technology will be useful in applications where inflammationreductions and/or tissue repair is necessary, but restrictions inanatomy would preclude the use of a material that will swell and/or takeup considerable space. For example, injections could be deliveredintraarticularly, intrathecally, intraoccularly, intracranially, or intopleural space.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a cross-sectional view of the wall of theurinary bladder (not drawn to scale). The following structures areshown: epithelial cell layer (A), basement membrane (B), tunica propria(C), muscularis mucosa (D), tunica submucosa (E), tunica muscularisexterna (F), tunica serosa (G), tunica mucosa (H), and the lumen of thebladder (L).

FIG. 2A is a photograph of lyophilized porcine urinary bladder matrix(UBM) sheet.

FIG. 2B is a photograph of lyophilized porcine urinary bladder matrix(UBM) powder.

FIG. 3 shows a qualitative observation of hydrogel formation followingterminal sterilization. Compared to the non-sterilized controls, theability of dermal ECM to form a hydrogel following terminalsterilization was abolished after the metallic ring molds (top image)were removed.

FIG. 4A shows non-sterilized lyophilized pre-gel.

FIG. 4B shows non-sterilized lyophilized pre-gel reconstituted in HCl

FIG. 4C shows non-sterilized lyophilized pre-gel reconstituted indeionized water.

FIG. 4D shows lyophilized pre-gel sterilized with EtO and reconstitutedeither in water or HCl. Comparison of FIGS. 4B-4C shows thatreconstitution was complete for all samples except those aftersterilization in HCl (FIG. 4D).

FIG. 5 shows sterilization of lyophilized SIS-ECM pre-gel results inhydrogel formation.

FIG. 6 shows sterilization of lyophilized UBM pre-gel results inhydrogel formation.

FIG. 7A is a comparison of various sterilization methods ofdecellularized UBM.

FIG. 7B is a qualitative comparison of FIG. 7A.

FIG. 8 is a graph showing chemotactic response of perivascular stemcells (PVSCs) to non-sterilized urinary bladder matrix (UBM) andsterilized (scCO₂, E-beam or gamma) UBM.

FIG. 9 is a graph showing TNFα production of human monocyte derivedmacrophages in response to treatment with UBM or sterilized (scCO₂,E-beam or gamma) UBM.

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of ranges is intendedas a continuous range including every value between the minimum andmaximum values. As used herein “a” and “an” refer to one or more.

As used herein, the term “comprising” is open-ended and may besynonymous with “including”, “containing”, or “characterized by”. Theterm “consisting essentially of” limits the scope of a claim to thespecified materials or steps and those that do not materially affect thebasic and novel characteristic(s) of the claimed invention. The term“consisting of” excludes any element, step, or ingredient not specifiedin the claim. As used herein, embodiments “comprising” one or morestated elements or steps also include, but are not limited toembodiments “consisting essentially of” and “consisting of” these statedelements or steps.

Methods are described herein of preparing extracellular matrix(ECM)-derived compositions comprising solubilized extracellular matrixobtained from any of a variety of tissues. Related compositions, devicesand methods of use also are described. The compositions are non-gellingwhen their temperature is raised to 37° C. According to one non-limitingembodiment, the ECM-derived composition is an injectable solution at 37°C. According to certain aspects, the composition is bioactive becausethe entire, intact ECM is solubilized and is not dialyzed, cross-linkedand/or otherwise treated to remove or otherwise inactivate ECMstructural or functional components, resulting in a highly bioactivecomposition. A general set of principles for preparing a non-gelling,solubilized ECM-derived composition is provided, along with specificprotocols for preparing such compositions. In comparison, non-limitingexamples of gelling (reverse-gelling) ECM-derived compositions aredescribed in U.S. Pat. No. 8,361,503, and United States PatentPublication Nos. 2010-0226895, and International Patent Publication Nos.WO 2011/087743 and WO 2013/009595.

As used herein, “sterilized”, “terminal sterilization”, or “terminallysterilized” refers to the complete, essentially complete, or practicallycomplete sterilization of a composition or device. This does not includedisinfection, e.g., with peracetic acid during preparation of an ECMmaterial as part of or ancillary to decellularization. As an example ofdisinfection, an ECM material can be disinfected by immersion in 0.1%(v/v) peracetic acid (a), 4% (v/v) ethanol, and 96% (v/v) sterile waterfor 2 h. The ECM material is then washed twice for 15 min with PBS(pH=7.4) and twice for 15 min with deionized water. Although this ischaracterized as disinfection, it is typically not acceptable undercurrent regulatory practice as a terminal sterilization method. Duringterminal sterilization, products are exposed to a validated process thatkills living microorganisms. In the context of ECM products,decellularized ECM material can be exposed to terminal sterilizationbefore solubilization, storage and/or commercial distribution. A varietyof methods for terminal sterilization are known in the art, includingexposure to: ethylene oxide, propylene oxide, gamma radiation, electronbeam radiation, gas plasma sterilization, and supercritical carbondioxide (see, e.g., White, A, et al., “Effective Terminal SterilizationUsing Supercritical Carbon Dioxide,” (2006) J. Biotech. 123(4):504-515).

The composition may also be disinfected by treatment withglutaraldehyde, which causes cross linking of the protein material, butthis treatment substantially alters the material such that it is slowlyresorbed or not resorbed at all and incites a different type of hostremodeling which more closely resembles scar tissue formation orencapsulation rather than constructive remodeling. Cross-linking of theprotein material can also be induced with carbodiimide or dehydrothermalor photooxidation methods. Cross-linked ECM material is not consideredto be a useful ECM material for purposes herein.

As indicated in the Examples below, the timing of terminal sterilizationsubstantially affects the ability of a digested, solubilized ECMmaterial to form a hydrogel by reverse gelling. Sterilization isperformed on a wet or dry solid sheet form or in comminuted powder formprior to enzymatic digestion with, e.g., an acid protease. By “dry” or“dried” it is meant dried or lyophilized to a point that essentially allwater is removed from a composition, recognizing that in practice, onemay not literally remove all water molecules from any composition. Thus“dry” or “dried” refers to a water content of, for example and withoutlimitation, less than 5.0, 1.0, 0.5, 0.1, 0.01, 0.001 or 0.0001% byweight of the composition (% wt.). Material can be dried by any process,such as, for example and without limitation, by simple evaporation atany non-damaging temperature, such as at room temperature, or bylyophilization (freeze drying).

According to one aspect of the invention, a method of preparing anon-gelling, solubilized ECM material is provided. In the method,decellularized or devitalized tissue, that is, extracellular matrix(ECM) material is terminally sterilized, for example by electron beam orgamma radiation, exposure to ethylene oxide gas or to supercriticalcarbon dioxide. It is then solubilized by digestion with an acidprotease, such as trypsin or pepsin, in an acidic solution to produce adigest solution. The digest solution optionally can be dried or isdried, for example by air drying or lyophilization. The composition canbe stored, packaged and/or distributed in this dried, e.g., lyophilized,state. The sterilized material is then hydrated, for instance bysolubilization in water or in an aqueous solution such as a TRIS bufferor PBS, or a salt solution such as a sodium chloride solution, such as(0.9%) saline to produce a sterilized digest solution. The sterilizeddigest solution is then brought to a pH between 7.2 and 7.8, e.g., 7.4,to produce a neutralized digest solution, for example, by mixing thesolution with an isotonic buffer or a base, such as, without limitationNaOH. The solution does not gel at 37° C., allowing the composition toremain as a solution at physiological temperatures. The rehydration andneutralization may be combined by rehydrating the dried, sterilizedcomposition in a buffer, such as PBS at pH 7.2-7.8, which willaccomplish the rehydration and neutralization step concurrently.

The compositions described herein find use as, without limitation, aninjectable graft (e.g., xenogeneic, allogeneic or autologous) fortissues, for example, bone or soft tissues, in need of repair oraugmentation most typically to correct trauma or disease-induced tissuedefects. Virtually any administration route is contemplated, includingtopical, enteral, and parenteral routes. The compositions also may beused as a lavage for rinsing external or internal, e.g., lumenal,surfaces, for therapeutic effect. For example, the composition may beused on the skin or on an external wound. The composition also can beadministered orally, nasally, by inhalation, enterally, intravaginally,etc. For example, injections could be delivered intraarticularly,intrathecally, intraoccularly, intracranially, and into pleural space.

The compositions may be implanted into a patient, human or animal, by anumber of methods. In one non-limiting embodiment, the compositions areinjected as a liquid into a desired site in the patient. As used herein,the term “seed,” “seeding,” or “seeded” refers to the addition,incorporation, propagation of, or spreading of a defined volume of acell suspension or a defined cell number into a specific composition.The composition may be pre-seeded with cells, and then preferablyinjected using a larger-bore, e.g. 16 gauge needle, to prevent shearingof cells.

As used herein, the terms “extracellular matrix” and “ECM” refer to anatural scaffolding for cell growth that is prepared bydecellularization of tissue found in multicellular organisms, such asmammals and humans. ECM can be further processed by, for instancedialysis or cross-linking. ECM is a complex mixture of structural andnon-structural biomolecules, including, but not limited to, collagens,elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials,chemoattractants, cytokines, and/or growth factors. In mammals, ECMoften comprises about 90% collagen, in its various forms. Thecomposition and structure of ECMs vary depending on the source of thetissue. For example, small intestine submucosa (SIS), urinary bladdermatrix (UBM) and liver stroma ECM each differ in their overall structureand composition due to the unique cellular niche needed for each tissue.

As used herein, the terms “intact extracellular matrix” and “intact ECM”refers to an extracellular matrix that retains activity of itsstructural and non-structural biomolecules, including, but not limitedto, collagens, elastins, laminins, glycosaminoglycans, proteoglycans,antimicrobials, chemoattractants, cytokines, and/or growth factors, suchas, without limitation comminuted ECM as described herein. The activityof the biomolecules within ECM can be removed chemically ormechanically, for example, by cross-linking and/or by dialyzing the ECM.ECM material useful for preparation of the non-gelling ECM materialdescribed herein essentially has not been cross-linked and/or dialyzed,meaning that the ECM has not been subjected to a dialysis and/or across-linking process. Thus, ECM that is cross-linked and/or dialyzed(in anything but a trivial manner which does not substantially affectthe functional characteristics of the ECM in its uses described herein)is not considered to be useful for the methods or compositions describedherein.

By “biocompatible”, it is meant that a device, scaffold composition,etc. is essentially, practically (for its intended use) and/orsubstantially non-toxic, non-injurious or non-inhibiting ornon-inhibitory to cells, tissues, organs, and/or organ systems thatwould come into contact with the device, scaffold, composition, etc.

An “ECM material,” is a material comprising or that is prepared from anextracellular matrix-containing tissue, and does not consist of asingle, isolated and purified ECM component, such as a purified collagenpreparation, as are commercially available. Any type of tissue-derivedmaterial can be used to produce the ECM materials in the methods,compositions and devices as described herein (see generally, U.S. Pat.Nos. 4,902,508; 4,956,178; 5,281,422; 5,352,463; 5,372,821; 5,554,389;5,573,784; 5,645,860; 5,711,969; 5,753,267; 5,762,966; 5,866,414;6,099,567; 6,485,723; 6,576,265; 6,579,538; 6,696,270; 6,783,776;6,793,939; 6,849,273; 6,852,339; 6,861,074; 6,887,495; 6,890,562;6,890,563; 6,890,564; and 6,893,666). In certain embodiments, the ECMmaterial is isolated from a vertebrate animal, for example and withoutlimitation, from a mammal, including, but not limited to, human, monkey,pig, cow and sheep. The ECM material can be prepared from any organ ortissue, including without limitation, urinary bladder, intestine, liver,esophagus and dermis.

In one embodiment, the ECM is isolated from a urinary bladder. Inanother embodiment, the ECM is isolated from intestine, or a portionthereof. The intestine extends from the pyloric sphincter to the anus,and includes: the small intestine, extending from the pyloric valve tothe ileocecal valve; the large intestine, extending from the ileocecalvalve; and portions thereof, including: the duodenum; the jejunum; theileum; the cecum; the appendix; the ascending, transverse, descendingand sigmoid colon; the rectum and/or the anal canal. The ECM may or maynot include the basement membrane portion of the ECM. In certainembodiments, the ECM includes at least a portion of the basementmembrane.

The type of ECM used in the scaffold can vary depending on the intendedcell types to be recruited during wound healing or tissue regeneration,the native tissue architecture of the tissue organ to be replaced, theavailability of the tissue source of ECM, or other factors that affectthe quality of the final scaffold and the possibility of manufacturingthe scaffold. For example and without limitation, the ECM may containboth a basement membrane surface and a non-basement membrane surface,which would be useful for promoting the reconstruction of tissue such asthe urinary bladder, esophagus, or blood vessel all of which have abasement membrane and non-basement membrane component.

In general, a method of preparing an ECM material as described hereinrequires the preparation of ECM material from an animal of a specificspecies, and typically from a specific organ. In certain aspects, theECM material is prepared from mammalian tissue. As used herein, the term“mammalian tissue” refers to tissue obtained from a mammal, whereintissue comprises any cellular component of an animal. For example andwithout limitation, tissue can be obtained from aggregates of cells, anorgan, portions of an organ, or combinations of organs. In certainaspects, the ECM material is prepared from tissue of a vertebrateanimal, for example and without limitation, human, monkey, pig, cattle,and sheep. In certain aspects, the ECM material is prepared from anytissue of an animal, for example and without limitation, urinarybladder, liver, CNS, adipose tissue, small intestine, large intestine,colon, esophagus, pancreas, dermis, and heart. In one aspect, the ECMmaterial is prepared from urinary bladder tissue, optionally excludingthe basement membrane portion of the tissue.

Following isolation of the tissue of interest, decellularization(devitalization) is performed by various methods, for example andwithout limitation, exposure to hypertonic saline, peracetic acid,Triton-X or other detergents. Decellularized tissue (ECM material) canthen be dried, either lyophilized (freeze-dried) or air dried. Dried ECMmaterial can be comminuted by methods including, but not limited to,tearing, milling, cutting, grinding, and shearing. The comminuted ECMmaterial can also be further processed into a powdered form by methods,for example and without limitation, such as grinding or milling in afrozen or freeze-dried state.

As used herein, the term “comminute” and any other word forms orcognates thereof, such as, without limitation, “comminution” and“comminuting”, refers to the process of reducing larger particles intosmaller particles, including, without limitation, by grinding, blending,shredding, slicing, milling, cutting, shredding. ECM can be comminutedwhile in any form, including, but not limited to, hydrated forms,frozen, air-dried, lyophilized, powdered, sheet-form.

As described above, the ECM material is terminally sterilized, andsolubilized. In order to prepare solubilized ECM material, comminutedECM material is digested with an acid protease in an acidic solution toform a digest solution. As used herein, the term “acid protease” refersto an enzyme that cleaves peptide bonds, wherein the enzyme hasincreased activity of cleaving peptide bonds in an acidic pH. Forexample and without limitation, acid proteases can include pepsin andtrypsin.

The digest solution of ECM material typically is kept at a constant stirfor a certain amount of time at room temperature. The ECM digest can beused immediately or be stored at −20° C. or frozen at, for example andwithout limitation, −20° C. or −80° C., and in the context of themethods and compositions described herein, dried and sterilized. Next,the pH of the digest solution is raised to a pH between 7.2 and 7.8 toproduce a neutralized digest solution. The pH can be raised by addingone or more of a base or an isotonic buffered solution, for example andwithout limitation, NaOH or PBS at pH 7.4. The method typically does notinclude a dialysis step, yielding a more-complete ECM-like composition.The composition retains more of the qualities of native ECM due toretention of many native soluble factors, such as, without limitation,cytokines.

As used herein, the term “isotonic buffered solution” refers to anisotonic solution that is buffered to a pH between 7.2 and 7.8 and thathas a balanced concentration of salts to promote an isotonicenvironment. As used herein, the term “base” refers to any compound or asolution of a compound with a pH greater than 7. For example and withoutlimitation, the base is an alkaline hydroxide or an aqueous solution ofan alkaline hydroxide. In certain embodiments, the base is NaOH or NaOHin PBS.

This neutralized digest solution can, at that point be incubated at asuitably warm temperature, for example and without limitation, at about37° C. The neutralized digest solution can be frozen and stored at, forexample and without limitation, −20° C. or −80° C. As used herein, theterm “neutralized digest solution” or “neutralized digest” refers to adigest or digest solution wherein the pH is increased, and can be in theform of a solution or dried composition. For example and withoutlimitation, a neutralized digest has a pH between 7.2 and 7.8.

In one aspect, an active agent or cells can be added to the solubilizedECM material as described herein. For example, cytokine, chemoattractantor cells can be mixed into the composition prior to use. For example andwithout limitation, useful active agents include growth factors,interferons, interleukins, chemokines, monokines, hormones, angiogenicfactors, drugs and antibiotics. Cells mixed into the solubilized ECMmaterial can be autologous or allogeneic with respect to the patient toreceive the composition/device comprising the composition. The cells canbe stem cells or other progenitor cells, or differentiated cells. In oneexample, a layer of dermis obtained from the patient is seeded for usein repairing damaged skin and/or underlying tissue. As used herein, theterm “active agent” refers to any compounds or compositions having apreventative or therapeutic effect, including and without limitation,antibiotics, peptides, hormones, organic molecules, vitamins,supplements, factors, proteins and chemoattractants.

As used herein, the terms “cell” and “cells” refer to any types of cellsfrom any animal, such as, without limitation, rat, mice, monkey, andhuman. For example and without limitation, cells can be progenitorcells, such as stem cells, or differentiated cells, such as endothelialcells, smooth muscle cells. In certain embodiments, cells for medicalprocedures can be obtained from the patient for autologous procedures orfrom other donors for allogeneic procedures.

The composition described herein can be used in multi-layered tissueconstructs, for example electrosprayed, e.g., onto or concurrently withan electrodeposited synthetic polymer composition, such as an elastomer.

In another embodiment of the pre-molded composition, the ECM compositionis contained within or absorbed into or adsorbed onto a laminar sheathof non-comminuted and non-digested ECM tissue, such as SIS or UBM, toadd physical strength to the composition. In this embodiment, sheets ofECM tissue, prepared in any manner known in the art, can be placed intoa mold prior, and the non-gelling composition is added to the mold. Thesheets of ECM tissue may be used as the mold, so long as they are formedand sewn or cross-linked into a desired shape. In this manner, a solidcomposition can be produced that has greater physical strength than isthe case of the composition described herein.

In another aspect, the solubilized ECM material is injected into apatient. The composition is injected at a locus in the patient where thematrix is needed for cell growth. For example and without limitation,where a patient has had tissue removed due to trauma, debridement and/orremoval of damaged, diseased or cancerous tissue, the composition can beinjected at the site of tissue removal to facilitate in-growth oftissue. The viscosity of the composition can be controlled by varyingthe amounts of water (e.g., by varying the amounts of water, acid, base,buffer (such as PBS) or other diluents) used to prepare the composition.In applications in which a small gauge needle is used, such as inendoscopy, a less viscous composition would be needed. In applicationsin which a larger gauge needle is available, a more viscous compositioncan be used. Also, use of a larger gauge needle, irrespective of theviscosity of the composition, favors mixing of live cells with thecomposition immediately prior to implantation with less risk of shearingthe cells.

In yet another aspect, the solubilized ECM material is contained in aspray device, for spraying onto tissue, a wound, etc.

In one aspect, the ECM material is directly injected into a patient. Inone embodiment, the composition is in a frozen state and is thawed andwarmed prior to injection.

In a further embodiment, a commercial kit is provided comprising acomposition described herein. A kit comprises suitable packagingmaterial and the composition. In one non-limiting embodiment, the kitcomprises a solubilized ECM material in a vessel, which may be thepackaging, or which may be contained within packaging. The compositionmay be frozen, cooled; e.g., kept at near-freezing temperatures, suchas, without limitation, below about 4° C. or kept at room temperature,e.g., 20-25° C., or even at physiological temperatures, for instance inpreparation for use. In another non-limiting embodiment, the kitcomprises a first vessel containing dried, e.g. lyophilized,non-gelling, solubilized ECM material as described herein. The vesselmay be a vial, syringe, tube or any other container suitable for storageand transfer in commercial distribution routes of the kit.

As used herein, the term “hybrid inorganic/ECM scaffold” refers to anECM material that is coated onto a biocompatible inorganic structure,such as, without limitation, a metal, an inorganic calcium compound suchas calcium hydroxide, calcium phosphate or calcium carbonate, or aceramic composition. In one embodiment, ultrasonication is used to aidin coating of the inorganic structure with the ECM-derived composition.As used herein, the term “ultrasonication” refers to the process ofexposing ultrasonic waves with a frequency higher than 15 kHz and lowerthan 400 kHz.

As used herein, the term “coat”, and related cognates such as “coated”and “coating,” refers to a process comprising of covering an inorganicstructure with the solubilized ECM material. For example and withoutlimitation, coating of an inorganic structure with a solubilized ECMmaterial as described herein includes methods such as pouring,embedding, layering, dipping, spraying.

In another aspect, the solubilized ECM material is coated onto abiocompatible structural material, such as a metal, an inorganic calciumcompound such as calcium hydroxide, calcium phosphate or calciumcarbonate, or a ceramic composition. Non-limiting examples of suitablemetals are cobalt-chrome alloys, stainless steel alloys, titaniumalloys, tantalum alloys, titanium-tantalum alloys, which can includeboth non-metallic and metallic components, such as molybdenum, tantalum,niobium, zirconium, iron, manganese, chromium, cobalt, nickel aluminumand lanthanum, including without limitation, CP Ti (commercially puretitanium) of various grades or Ti 6Al 4V (90% wt. Ti, 6% wt. Al and 4%wt. V), stainless steel 316, Nitinol (Nickel-titanium alloy), titaniumalloys coated with hydroxyapatite. Metals are useful due to highstrength, flexibility, and biocompatibility. Metals also can be formedinto complex shapes and many can withstand corrosion in the biologicalenvironments, reduce wear, and not cause damage to tissues. In onenon-limiting example, the metal is femoral or acetabular component usedfor hip repair. In another example, the metal is a fiber or otherprotuberance used in permanent attachment of a prosthesis to a patient.Other compositions, including ceramics, calcium compounds, such as,without limitation, aragonite, may be preferred, for example and withoutlimitation, in repair of or re-shaping of skeletal or dental structures.Combinations of metal, ceramics and/or other materials also may proveuseful. For instance, a metal femoral component of a hip replacement maycomprise a ceramic ball and/or may comprise a plastic coating on theball surface, as might an acetabular component.

Metals, as well as other materials, as is appropriate, can be useful inits different forms, including but not limited to wires, foils, beads,rods and powders, including nanocrystalline powder. The composition andsurface of metals or other materials can also be altered to ensurebiocompatibility, such as surface passivation through silane treatments,coating with biocompatible plastics or ceramics, composite metal/ceramicmaterials. The materials and methods for their employment are well-knownin the field of the present invention.

A difficulty with using metal inserts to repair a patient's skeletalstructure is that the inserts must be anchored/attached to existingskeletal parts. Traditional methods employ cement and/or screws. In thecase of prostheses, the prostheses are not connected to a patient'stissue except, typically, by cementing. Therefore, it is desirable tobiologically attach a patient's tissue to a medical device. This may beaccomplished by coating surfaces of the implant with the solubilized ECMmaterial described herein, which will facilitate in-growth of tissue andthus attachment of the device. A variety of porous structures can beattached to the implant to create a scaffold into which the solubilizedECM material, and later cells or other tissue (e.g., bone) caninfiltrate. Structures include, without limitation: woven or non-wovenmesh, sponge-like porous materials, fused beads, etc. The porousscaffold will facilitate formation of a strong bond between livingtissue, including bone, and the device. The “pores” of the porousscaffold may be of any size that will permit infiltration of asolubilized ECM material, optionally facilitated by ultrasound or othertreatments that would assist in permeation of the composition, and latercells or other biological materials, such as bone, cartilage, tendons,ligaments, fascia or other connective tissue, into the scaffolding. Inone aspect, metal fibers are attached to the device, and the metalfibers are coated with a solubilized ECM material described herein,thereby permitting in-growth of tissue within the fibers. In a secondembodiment, a matrix of small beads is welded or otherwise attached to asurface of the device and a solubilized ECM material described herein iscoated onto the bead matrix, facilitating in-growth of tissue among thebeads. In one example, a device contains a protuberance of fibers, whichcan be inserted inside a bone, permitting fusion of the metal fiberswith the bone. In one aspect, the solubilized ECM material is seeded andincubated with a suitable cell population, such as autologousosteoblasts, to facilitate bone in-growth.

A device (e.g., a prosthesis) as described herein can be coated with thedescribed ECM composition. In another embodiment, the composition isapplied to the device or prostheses. The composition on the device canthen be dried, e.g. lyophilized and the entire device can be terminallysterilized, followed by packaging and distribution. The lyophilizedproduct on the device can be hydrated by an end-user.

In use, the device which is coated with a suitable scaffolding and ECMcomposition as described herein may be contacted with cells, e.g. of apatient or allogeneic cells, and the cells are allowed to infiltrate thematrix. The in-growth or infiltration of cells can occur in vivo or exvivo, depending on optimization of methods. For example and withoutlimitation, in the case of a femoral implant, the implant can beinserted into the femur and cells of a patient, and desirable bonetissue, infiltrates the scaffolding to fuse the device to the bone. Inanother embodiment, for example in the case of an artificial tendon orligament, a biopsy of a patient's tendons or ligaments is incubated withan appropriate scaffold in order to create an autologous ligament ortendon graft.

EXAMPLES Example 1—Preparation of Porcine Extracellular Matrix (ECM)(UBM)

The preparation of UBM has been previously described (Sarikaya A, et al.Tissue Eng. 2002 February; 8(1):63-71; Ringel R L, et al. J Speech LangHear Res. 2006 February; 49(1):194-208). In brief, porcine urinarybladders were harvested from 6-month-old 108-118 kg pigs(Whiteshire-Hamroc, IN) immediately following euthanasia. Connectivetissue and adipose tissue were removed from the serosal surface and anyresidual urine was removed by repeated washes with tap water. The tunicaserosa, tunica muscularis externa, the tunica submucosa, and majority ofthe tunica muscularis mucosa were mechanically removed. The urothelialcells of the tunica mucosa were dissociated from the luminal surface bysoaking the tissue in 1.0 N saline solution yielding a biomaterialcomposed of the basement membrane plus the subjacent tunica propria,which is referred to as urinary bladder matrix (UBM). See FIG. 1 forcross-sectional view of the wall of the urinary bladder, as well asstructures included within.

The UBM sheets were disinfected for two hours on a shaker in a solutioncontaining 0.1% (v/v) peracetic acid, 4% (v/v) ethanol, and 95.9% (v/v)sterile water. The UBM sheets (as in FIG. 2A) were then lyophilized(FIG. 2B) using a FTS Systems Bulk Freeze Dryer Model 8-54 and powderedusing a Wiley Mini Mill.

Example 2—Preparation of Porcine Spleen ECM

Fresh spleen tissue was obtained. Outer layers of the spleen membranewere removed by slicing, where remaining tissue was cut into uniformpieces. Remnants of outer membrane were trimmed, then rinsed three timesin water. Water was strained by using a sieve. Splenocytes were lysed bymassaging. Spleen slices were incubated in a solution of 0.02%trypsin/0.05% EDTA at 37° C. for 1 hour in a water bath. If necessary,splenocytes were further lysed by massaging. After rinsing, slices weresoaked in 3% Triton X-100 solution and put on a shaker for 1 hour. Ifnecessary, splenocytes were further lysed by massaging. Slices were thensoaked in 4% deoxycholic acid solution and put on a shaker for 1 hour.After thoroughly rinsing, the purified spleen ECM was stored for furtherprocessing. This tissue was disinfected and dried.

Example 3—Preparation of Porcine Liver Stroma ECM

Fresh liver tissue was obtained. Excess fat and tissue were trimmed.Outer layers of the liver membrane were removed by slicing, whereremaining tissue was cut into uniform pieces. Remnants of outer membranewere trimmed using a scalpel or razor blade, then rinsed three times inwater. Water was strained by using a sieve. Cells were lysed bymassaging. Liver slices were incubated in a solution of 0.02%trypsin/0.05% EDTA at 37° C. for 1 hour in a water bath. If necessary,cells were further lysed by massaging. After rinsing, slices were soakedin 3% Triton X-100 solution and put on a shaker for 1 hour. Ifnecessary, cells were further lysed by massaging. Slices were thensoaked in 4% deoxycholic acid solution and put on a shaker for 1 hour.After thoroughly rinsing, the purified liver stroma was stored indeionized water for further processing. This tissue was next disinfectedwith peracetic acid treatment and dried.

Example 4—Preparation of Human Liver Stroma ECM

Fresh liver tissue was obtained. Excess fat and tissue were trimmed.Outer layers of the liver membrane were removed by slicing, whereremaining tissue was cut into uniform pieces. Remnants of outer membranewere trimmed using a scalpel or razor blade, then rinsed three times inwater. Water was strained by using a sieve. Cells were lysed bymassaging. Liver slices were incubated in a solution of 0.02%trypsin/0.05% EDTA at 37° C. for 1 hour in a water bath. If necessary,cells were further lysed by massaging. After rinsing, slices were soakedin 3% Triton X-100 solution and put on a shaker for 1 hour. Ifnecessary, cells were further lysed by massaging. Slices were thensoaked in 4% deoxycholic acid solution and put on a shaker for 1 hour.After thoroughly rinsing, the purified liver stroma was stored indeionized water for further processing. This tissue was next disinfectedwith peracetic acid treatment and dried.

Example 5—Preparation of Porcine Ovarian ECM

Fresh ovarian tissue is obtained within 6 hours of harvest. Ovaries wereremoved and stored in physiological saline tissue until ready fordissection and residual uterine tissue was removed. Longitudinalincisions were made through the hilum of the ovary and the follicleswere disrupted. Once all the follicles have been disrupted, the ECM hasbeen harvested from the ovaries. Rinse three times in filtered water andstrain the water using a sieve. Cells were lysed by gentle massaging.ECM was incubated in a solution of 0.02% trypsin/0.05% EDTA at 37° C.for 1 hour in a water bath and then rinsed. If necessary, cells werefurther lysed by massaging. ECM was soaked in 3% Triton X-100 solutionand put on a shaker for 1 hour. After rinsing, cells were further lysedby massaging if necessary. Slices were then soaked in 4% deoxycholicacid solution and put on a shaker for 1 hour. After thoroughly rinsingto remove residual surfactant, the ECM was stored in sterile/filteredwater until further use. This tissue was next disinfected with peraceticacid treatment and dried.

Example 6: Preparation of Spinal Cord and Dura Mater ECM

Using forceps, scissors and a scalpel, dura mater was removed fromporcine spinal cord. The inner dura mater surface was scrapped withscalpel blade to remove any debris. The spinal cord and dura were placedin separate containers and treated in the same manner as below. Thespinal cord was cut either longitudinally or in cross-section toincrease surface area and placed in a cassette. Optionally tissue wasenzymatically treated using trypsin-EDTA for 30 minutes at 37° C. Thetissue was incubated in Triton X-100™ (4-octylphenol polyethoxylate)solutions at 3% for periods up to 2-3 days at 4° C. This step wasrepeated with a solution of Triton X-100™ at 6% and again with asolution of Triton X-100™ at 9%. The spinal cord tissue was incubated inlecithin or lecithin-deoxycholate to remove lipids overnight at 4° C.Dura mater was not subjected to this procedure. Tissue was then washedin Triton X-100™ 3% or SDS 1% for 1-2 hours. The tissue was rinsed inPBS 3X for 15 minutes at room temperature. Then the tissue was incubatedin a solution of DNase I for 1 hour at room temperature. The tissue waswashed in PBS three times for 15 minutes at room temperature. Lastly,the tissue was washed in deionized water three times for 15 minutes atroom temperature. The procedure produced a gel-like acellular spinalcord material, and a sheet of acellular dura mater material.

Example 7—Preparation of Adipose ECM

Frozen adipose tissue was thawed in water and manually massaged tohasten the lysis of cells. Tissue was placed into a flask containing0.02% trypsin/0.05% EDTA solution and incubated at 37° C. for one hourthen rinsed briefly in distilled deionized water (ddH₂O) and manuallymassaged again. Tissue was then placed into a flask containing 3% TritonX-100 and placed on an orbital shaker for 1 hour at room temperature.Following a brief water rinse, tissue was placed into a 4% deoxycholicacid solution and again placed on an orbital shaker for 1 hour at roomtemperature. Tissue was rinsed three times in water and stored at 4° C.overnight. The tissue was then subjected to a 4% ethanol and 0.1%peracetic acid solution on an orbital shaker for 2 hours at followed bytwo phosphate buffered saline (PBS, pH 7.4) and two water washes of 15minutes each at room temperature. The resulting material was then washedin 100% n-propanol for one hour on an orbital shaker at room temperatureand washed in four changes of ddH₂O for one hour to remove then-propanol prior to lyophilization.

Example 8—Preparation of Neural-Derived ECM Gel

Murine spinal cord tissue was stored at −80° C. until needed for ECMderivation processing. The material was thawed and the dura mater wasremoved from the spinal cord, and the spinal cord was then cut intoquarters longitudinally of about 1 inch length and uniform thickness.The spinal cord pieces were placed into water overnight at 4° C. and 120rpm to mechanically-disrupt the native tissue architecture prior todecellularization. After about 18 h the spinal cord pieces were removedfrom the water by straining onto a mesh or sieve with hole size of about840 μm. The pieces of spinal cord were collected with forceps and placedinto a flask for protease digestion with 0.02% trypsin/0.05% EDTAsolution. The digestion was allowed to proceed in a water bath for 1 hat 37° C. while shaking at 120 rpm. After one hour, the solution wasstrained off and spinal cord tissue was rinsed gently under a stream ofwater, detangling as required. The spinal cord pieces were returned tothe flask, collecting as many smaller tissue pieces as possible from thestrainer using forceps. 3% Triton X-100 solution was then added to theflask to begin decellularization of the tissue, which was placed on ashaker for 1 h at 200 rpm. After one hour the tissue was strained,rinsed, and collected. The tissue pieces were placed back into theflask, and then were subjected to osmotic shock for additionaldecellularization. Hypertonic 1 M sucrose was added to the flask andplaced on a shaker for 15 min at 200 rpm. The tissue was strained,rinsed, collected and combined with hypotonic solution (deionized water)and placed on shaker for 15 min at 200 rpm, to lyse any remaining cells.The decellularized tissue was again strained, rinsed, and reclaimed intoa flask. 4% deoxycholate solution was added to the flask and placed upona shaker for 1 h at 200 rpm. Subsequently, the tissue pieces werestrained and rinsed repeatedly in type I (ultrapure) water until alltraces of surfactants (bubbles) were removed. The remaining tissue, nowenriched into ECM, was collected and disinfected using a peracetic acidsolution (made up of Type I water (96%) and 100% EtOH alcohol (4%)) at aratio of 20:1 peracetic acid solution to weight of ECM, and shaken at200 rpm for two hours. Following a series of rinse steps in PhosphateBuffered Saline (PBS), the ECM was frozen at −20° C. and thenlyophilized until all water was removed.

Example 9—Sterilization of Pre-Digest ECM Materials

It has been noted that terminal sterilization of an ECM scaffoldinhibits subsequent hydrogel formation of protease-solubilized ECMmaterials. The following illustrates the effect of sterilization onqualitative hydrogel formation, using dermal ECM that has not beendialyzed (that is, intact ECM as described above). Porcine dermis wasdecellularized essentially as described above, and subjected to variousmethods of terminal sterilization. The dermal ECM sheets were terminallysterilized by exposure to (1) gamma radiation at a dosage of 10 kGy, 25kGy, and 40 kGy, (2) electron beam radiation at a dosage of 10 kGy, 25kGy, and 40 kGy, and (3) ethylene oxide (EtO) gas at a dose of 750 mg/hfor 16 h. Control dermal ECM sheets were not sterilized. The sterilizedand control dermal ECM was then mechanically comminuted in a Wiley Millwith a 60 mesh sieve, enzymatically digested for 48 hours at 4° C. in 1mg/ml pepsin in a solution of 0.01 N HCl to produce a pre-gel, andtested for hydrogel formation by neutralization to approximately pH 7.4at room temperature (20-25° C.) and then raising the temperature to 37°C. in an incubator without CO₂. FIG. 3 shows the terminally sterilizeddermal ECM were unable to form a hydrogel regardless of sterilizationmethod whereas the non-sterilized dermal ECM formed a solid gel andmaintained form after removal of metallic ring molds.

Example 10—Sterilization of Lyophilized Product

Due to the ineffective hydrogel formation after terminal sterilizationof the pre-digest decellularized ECM material, and recognizing theclinical/commercial need for sterilization, we hypothesized thatchanging the form of material prior to sterilization would allow for ECMgelation. Instead of the lyophilized solid ECM sheets, we sterilized alyophilized pre-gel prepared from non-dialyzed (intact ECM as describedabove), mechanically comminuted ECM that is enzymatically digestedessentially as described above) and tested whether hydrogel formationwould occur. ECM derived from small intestinal submucosa (SIS-ECM) wasdecellularized according to a standard protocol without dialysis. TheSIS-ECM powder was enzymatically digested in the same manner as above,frozen at −80 C on dry ice, and lyophilized. The lyophilized pre-gel wasthen sterilized by exposure to EtO gas at a dose of 750 mg/h for 16 hand/or reconstituted in deionized water or 0.01N HCl (FIGS. 6A-6D).FIGS. 6A-6D show complete reconstitution at room temperature using waterand HCl prior to sterilization but after sterilization (FIG. 4D) onlypre-gel in deioinized water was completely reconstituted.

Since only pre-gel in deionized water was completely reconstituted,these samples were tested for hydrogel formation. FIG. 5 shows that bothnon-sterilized and EtO sterilized pre-gel resulted in a solid gel whenneutralized and placed in a 37° C. incubator without CO₂, whichmaintained form after removal of metallic ring molds.

To corroborate the results shown in FIG. 5, ECM derived from urinarybladder (UBM) was decellularized according to a standard protocol andtested in the same manner as the SIS-ECM, above. The results similarlyshowed that sterilization of the lyophilized pre-gel was conducive tohydrogel formation (FIG. 6).

Example 11—Sterilization of Lyophilized Product—Further Studies

Expanding the work of the Examples above, ECM materials, preparedessentially as described above, are sterilized in different forms, asfollows: ECM material that is not digested, both in powder and 2D sheetform; hydrated powder or 2D sheet ECM material that is not digested;pre-gel solution that is acid-protease digested, but not neutralized;and/or lyophilized pre-gel that is digested, but not neutralized. ECMmaterial from various sources are tested, including urinary bladder,spleen, liver, heart, pancreas, ovary, small intestine, central nervoussystem (CNS), adipose tissue, and/or bone.

The materials are sterilized by ethylene oxide, gamma radiation (2 kGy,30 kGy @ ambient and −80° C.), electron beam radiation (2 kGy, 30 kGy @ambient and −80 C), and/or supercritical CO₂ (low and/or high). Anon-sterilized control also is run.

Example 12—Decellularized Urinary Bladder Matrix (UBM) Subjected toVarious Sterilization Methods

Urinary bladder matrix was decellularized according to a standardprotocol and subjected to various sterilization methods. Hydrogelformation was tested following sterilization of (1) the UBM sheet, (2)mechanically comminuted UBM powder, and (3) following digestion andlyophilization. Each gel was formed in a ring mold and imaged (FIG. 7A).Hydrogel formation was then tested with the most rigid hydrogels formedby the lyophilized digests, followed by the powder form, as shown by thestorage modulus values (see FIG. 7B). Sterilization of the UBM sheet didnot allow for subsequent hydrogel formation. Each sterilization methodhas an impact upon hydrogel formation but this can be mitigated by thestarting form of the ECM material, most clearly evidenced by acomparison of the storage moduli of the scCO₂ and EtO lyophilized digestversus powder forms. Interestingly, the non-sterilized control samplesalso show a distinct difference in hydrogel formation with thelyophilized digest forming a more rigid gel. Qualitatively, thelyophilized digest formed the most rigid hydrogel followed by powderwhile sterilization of the UBM sheet did not allow for subsequenthydrogel formation. The qualitative trends in gel rigidity werecorroborated by rheological characterization of the gel stiffness (i.e.,storage modulus or G′). Together, these Figures highlight the importanceof the form of ECM (i.e., sheet, powder, or digest) that is exposed tosterilization.

Example 13—Preparation of Non-Gelling Solubilized ECM

1—Decellularize a tissue of interest (e.g., small intestine, urinarybladder, liver, bone, etc.)

2-Sterilize the ECM with a method of terminal sterilization (e.g.,exposure to ethylene oxide or ionizing radiation). Sterilization can beperformed on ECM in dry or hydrated forms and in sheet or powder forms.

3-After sterilization, mechanically comminute and solubilize the ECM byenzymatic digestion (e.g., with pepsin).

4-Neutralize the salt concentration and pH of the solubilized ECM andthe soluble, non-gelling ECM is ready for use.

Example 14—Use of Non-Gelling Solubilized ECM for Treatment ofRheumatoid Arthritis

The technology could be used to treat individuals with rheumatoidarthritis, for example, or other inflammatory joint conditions. Whereascurrent treatment is intraarticular injection of a corticosteroid, whichis associated with poor side-effects, a soluble extracellular matrix(ECM) prepared as described herein, would act to reduce localinflammation. Particularly in the intraarticular space it is importantthat the ECM be soluble but non-gelling so there is not a physicalobstruction to articular movement.

Example 15—Evaluation of Non-Gelling Solubilized ECM

Urinary Bladder Matrix was prepared by accepted protocol: mechanicaldelamination, washed in 0.1% peracetic acid, and rinsed in saline andwater, essentially as described herein. The material was thenlyophilized and separated into groups based on sterilization treatmentof 30 kGy gamma irradiation, 30 kGy electron beam irradiation, andsupercritical CO₂ sterilization. Following sterilization, each scaffoldwas ground to particulate and solubilized in a pepsin buffer for 24hours. The pH and salt concentration in the digest was then balancedessentially as described herein for preparation of an ECM hydrogel, andlack of gelation was validated by warming to 37° C. prior to use inexperiments. Sterilization in this manner prevented gelation independentof choice of sterilization method

FIG. 8 shows chemotactic response of perivascular stem cells (PVSCs) tonon-sterilized urinary bladder matrix (UBM) and sterilized (scCO₂,E-beam or gamma) UBM. Chemotaxis assays were performed in standardchambers with 8 μm filters (Neuro Probe, Gaithersburg, Md.) coated withrat-tail collagen (BD Biosciences, San Jose, Calif.). Solubilizedsterilized and non-sterilized UBM samples were loaded into the bottomwells of the chamber and the collagen coated filter was placed on top.PVSCs were grown to 90% confluence before overnight incubation in DMEMwith 0.5% heat inactivated FBS. Cells were trypsinized and resuspendedin DMEM and 30,000 cells were loaded into the top well of the chemotaxischamber, separated from the bottom by the filter. Chambers were placedinto a humidified atmosphere at 37° C. with 5% CO₂ for three hours.Migrated cells were stained with DAPI (Fisher Scientific, Waltham,Mass.), imaged using a Zeiss Axio-Observer Z.1 microscope (Oberkochen,Germany) with 10× objective and quantified with ImageJ (NIH, Bethesda,Md.). Changes in chemotactic response for non-sterilized and sterilizedUBM were expressed as a fold change compared to migration media control.There was no significant difference in the chemotactic response of PVSCsto UBM or sterilized UBM.

FIG. 9 shows TNFα production of human monocyte derived macrophages inresponse to treatment with UBM or sterilized (scCO₂, E-beam or gamma)UBM. Human monocytes (THP-1 ATCC TIB-202) were exposed to 320 nM phorbol12-myristate (PMA) for 24 hours to induce differentiation intomacrophages. Adherent macrophages were washed in PBS and placed in freshmedia for 48 hours and then treated with solubilized non-sterilized orsterilized UBM for 48 hours. Culture supernatants were centrifuged topellet the cells and debris; supernatants were harvested and frozen at−80° C. Quantification of TNFα production was undertaken using acommercially available ELISA kit (BD Bioscience, San Jose, Calif.) andTNFα production was normalized to PBS treatment control. There was nosignificant difference in TFNα production when macrophages were exposedto UBM or sterilized UBM.

The present invention has been described in accordance with severalexamples, which are intended to be illustrative, rather than limiting,in all aspects. Thus, the present invention is capable of manyvariations in detailed implementation, which may be derived from thedescription contained herein by a person of ordinary skill in the art,and should not be limited by the preceding description, but should beconstrued to be as broad in scope as the following claims.

1-45. (canceled)
 46. A composition comprising a solution comprisingdecellularized, solubilized, terminally sterilized, intact extracellularmatrix (ECM) that has not been dialyzed or cross-linked, and an acidprotease, the solution having a pH in the range of 7.2 to 7.8, whereinthe solution does not form a hydrogel when the temperature of thesolution is raised to 37° C.
 47. The composition of claim 46, whereinthe acid protease is pepsin or trypsin.
 48. The composition of claim 46,wherein the ECM is derived from a tissue selected from urinary bladder,spleen, liver, heart, pancreas, ovary, small intestine, large intestine,colon, central nervous system tissue, adipose tissue, bone, esophagus,or dermis.
 49. The composition of claim 46, wherein the ECM is derivedfrom small intestinal submucosa (SIS) or urinary bladder matrix (UBM).50. The composition of claim 46, wherein the pH is in the range of 7.2to 7.4.
 51. The composition of claim 46, wherein the composition is aninjectable solution at 37° C.
 52. The composition of claim 46, whereinthe composition is lyophilized.
 53. The composition of claim 46, whereinthe composition is contained within, absorbed into, or adsorbed onto alaminar sheath of non-comminuted and non-digested ECM.
 54. Thecomposition of claim 46, wherein the composition further comprises acell.
 55. The composition of claim 46, wherein the composition furthercomprises a chemoattractant, a cytokine, or an antibiotic.
 56. Thecomposition of claim 46, wherein the ECM is mammalian ECM.
 57. Thecomposition of claim 56, wherein the mammalian ECM is selected fromhuman, monkey, pig, cow, or sheep ECM.
 58. A composition comprising: anacidic solution comprising an acid protease and decellularized,solubilized, terminally sterilized, intact extracellular matrix (ECM)that has not been dialyzed or cross-linked, wherein the acidic solution,once neutralized, does not form a hydrogel when the temperature israised to 37° C.
 59. The composition of claim 58, wherein thecomposition, when neutralized, has a pH of 7.2 to 7.8.
 60. Thecomposition of claim 58, wherein the composition, when neutralized, hasa pH of 7.2 to 7.4.
 61. The composition of claim 60, wherein the acidprotease is pepsin or trypsin.
 62. The composition of claim 58, whereinthe ECM is derived from a tissue selected from urinary bladder, spleen,liver, heart, pancreas, ovary, small intestine, large intestine, colon,central nervous system tissue, adipose tissue, bone, esophagus, ordermis.
 63. The composition of claim 58, wherein the ECM is derived fromsmall intestinal submucosa (SIS) or urinary bladder matrix (UBM). 64.The composition of claim 58, wherein the ECM is mammalian ECM.
 65. Thecomposition of claim 64, wherein the mammalian ECM is selected fromhuman, monkey, pig, cow, or sheep ECM.